Project Summary/Abstract Traumatic brain injury (TBI) is a serious health problem that affects approximately 1.5 million people in the United States each year and causes serious cognitive and physical deficits; the majority of TBI's fall within the mild (mTBI) category. Repeated mild TBI is a significant problem due to the risk of worsening outcome. Impaired glucose uptake in the brain is correlated with functional impairments, and our previous work has shown an acute but transient depression in 18fluorodeoxyglucose (FDG) uptake after mild TBI in a rodent model using positron emission tomography (PET) imaging. We have also shown that repeating a mild injury during this period of depressed glucose uptake resulted in marked worsening of functional and histological outcomes. Preliminary data from our laboratory in a moderate TBI model has shown that intranasal insulin significantly increases glucose uptake in the hippocampus, cortex and amygdala and induces significant improvements in cognitive function. In addition, intranasal insulin administration shifts microglial polarization to the M2, or anti-inflammatory and protective, phenotype. However, it is currently unclear if intranasal insulin is equally effective in the mild TBI model, and the mechanism of action has not been clarified. Therefore, we propose to investigate the hypothesis that intranasal insulin increases neuronal viability and reduces microglial-related inflammation, improving outcome following single mild TBI and reducing vulnerability to repeated mild TBI. To test this hypothesis, we propose two specific aims. The first aim will establish the mechanism of insulin's actions on neurons and microglia after mild TBI. In this aim, we will assess the effect of intranasally administered insulin on the viability and activation of neurons, astrocytes and microglia after mTBI and the association of these changes to glucose uptake after injury. The second aim will demonstrate that acute administration of intranasal insulin improves recovery and reduces vulnerability to a repeated impact following an mTBI. In this aim we will administer intranasal insulin after an mTBI and assess histological and functional recovery in animals after single injury or after repeated injury. This proposal will increase the understanding of the mechanisms behind the increase in vulnerability to worsened outcome following a second mild TBI. In addition, this proposal will assess intranasal insulin as a therapy to reduce negative impacts of a single and repeated mTBI, extending our previous discoveries into the efficacy of intranasal insulin in moderate contusion TBI into the mild and repeated mild TBI field, and providing an exploration of the mechanism of this therapy, which will be essential to the development of future clinical trials.